J Clin Exp Hematop Vol. 54,No. 3,Dec. 2014

Case Study B-Cell Prolymphocytic Carrying t(8;14)(q24;q32), Associated with Both Autoimmune Hemolytic Anemia and Pure Red Cell Aplasia

Futoshi Iioka,1) Takashi Akasaka,1) Masahiko Hayashida,2) Atsuko Okumura,2) and Hitoshi Ohno1,2)

An 80-year-old man was referred to our department because of . His white cell count was 17.1 × 103/µL, with 64% prolymphocytes. He did not exhibit splenomegaly or lymphadenopathy. Prolymphocytes were CD5+,CD10-,CD19+, CD20+,CD21+weak,CD22+,CD23-, and HLA-DR+, and expressed µd/l cell-surface immunoglobulins. G-banding and fluores- cence in situ hybridization using c-MYC and immunoglobulin heavy-chain (IgH) gene probe revealed that leukemia cells carried the t(8;14)(q24;q32)/c-MYC-IgH fusion gene, and breakage and reunion occurred within the non-coding region of c-MYC exon 1 as well as the a switch region of IgH. Nine months after the initial presentation, the patient’s hemoglobin level fell to 5.7 g/dL. Coombs’ test was positive and marked hypoplasia oferythroid precursors was detected in his bone marrow. The patient was treated with prednisolone followedby 4 weekly doses ofrituximab, which led to resolution ofthe anemia and complete response ofthe underlying leukemia. The role oft(8;14)(q24;q32)/c- MYC-IgH in the pathogenesis ofB-cell prolymphocytic leukemia (B- PLL) may not be identical to that in aggressive lymphoid neoplasms, and, in the present case, autoantibodies targeting both mature red cells and erythroid precursors may have been concurrently produced in the setting ofB-PLL. 〔J Clin Exp Hematop 54(3) : 219-224, 2014〕

Keywords: B-cell prolymphocytic leukemia, t(8;14)(q24;q32)/c-MYC-IgH, autoimmune hemolytic anemia, pure red cell apla- sia, rituximab

14)(q13;q32) and its molecular equivalent indicates the leuke- INTRODUCTION mic manifestationofmantle cell lymphoma instead ofB- B-cell prolymphocytic leukemia (B-PLL) is a rare lym- PLL.1-3 phoid neoplasm that is characterized by the proliferation of Immune-mediated anemia is often associated with lym- prolymphocytes with the B-cell phenotype in the peripheral phoid neoplasms at variable frequencies.4 Autoimmune he- , bone marrow, and spleen.1 Most patients present with molytic anemia (AIHA) is the most common autoimmune a marked increase in white cell counts and splenomegaly, and complication ofB-CLL, with a frequencyof5 to 10%. 5 On respond poorly to the standard treatments for B-cell chronic the other hand, sporadic case reports and small case series of lymphocytic leukemia (B-CLL).1-3 In addition to its morpho- lymphoid neoplasms associated with pure red cell aplasia logical and clinical features, the differential diagnosis of B- (PRCA) are found in the literature.4,6,7 The mechanisms re- PLL from other B-cell /lymphomas requires immu- sponsible for the two types of anemia may be attributable to nophenotypic and cytogenetic studies. In contrast to B-CLL the production ofautoantibodies against red cells or their cells, B-PLL cells strongly express surface immunoglobulin precursors by neoplastic cells themselves or polyclonal B (µ ± d) and pan-B markers (CD19, CD20, CD22, and CD79a), cells stimulated by the underlying neoplasm. and the positivity ofCD5 and CD23 was previously shown to We here describe a case ofB-PLL, the leukemia cells of be 20-30% and 10-20%, respectively.1 The presence oft(11; which carried t(8;14)(q24;q32). This disease was associated with progressive anemia and finally with the aplastic crisis of Received: July 10, 2014 red cell precursors in the bone marrow. The morphology, Revised : September 3, 2014 immunophenotype, and cytogenetic abnormalities ofleukemia Accepted: September 30, 2014 cells as well as their response to treatments with prednisolone 1)Department ofHematology, Tenri Hospital, Tenri, Nara, Japan and rituximab are described. 2)Tenri Institute ofMedical Research, Tenri, Nara, Japan Corresponding author: Futoshi Iioka, M.D., Department ofHematology, Tenri Hospital, 200 Mishima, Tenri, Nara 632-8552, Japan E-mail: [email protected]

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chain (IgH) gene dual fusion probe (Vysis IGH/MYC/CEP8 CASE REPORT Tri-Color DF FISH Probe Kit, Abbott Laboratories), and An 80-year-old man was referred to our department be- found that 54.8% of the nuclei contained two yellow signals cause oflymphocytosis fora duration ofover two years. He indicative ofthe c- MYC-IgH fusion gene (Fig. 3A). We then was asymptomatic with no hepatosplenomegaly or superficial applied the probe to the chromosome preparation and identi- lymphadenopathy. His hemoglobin level was 13.3 g/dL, pla- fied the der(8) and der(14) chromosomes involved in t(8;14) telet count 175 × 103/µL, and white cell count 17.1 × 103/µL. (q24;q32) (Fig. 3B). The other c-MYC signal was localized His white cells included 64% prolymphocytes with moder- on the end ofthe marker chromosome, which was determined ately condensed nuclear chromatin, prominent nucleoli, and a to be der(3)t(3;8)(p13;q13) (Fig. 3B), or on normal chromo- faintly basophilic cytoplasm (Fig. 1A). His blood chemistry some 8. The complete karyotype ofleukemia cells according was unremarkable, except for a lactate dehydrogenase level of to the ISCN8 was 47,XY,+der(3)t(3;8)(p13;q13),-8,t(8;14) 315 IU/L. His soluble interleukin-2 receptor level was 2,480 (q24;q32),+18[5]/48,XY,+add(3)(p13),t(8;14)(q24;q32),+18 U/mL (normal range, 135-483 U/mL). [2]/46,XY[4] (Fig. 4). A bone marrow aspirate smear showed 12.0% erythroid To confirm t(8;14)(q24;q32)/c-MYC-IgH, high-molecular- lineage cells, 13.8% myeloid lineage cells, and 69.1% pro- weight genomic DNA extracted from the peripheral blood (Fig. 1B); megakaryocytes appeared normal. was subjected to a long-distance polymerase chain reaction Prolymphocytes were CD5+,CD10-,CD19+,CD20+, (LD-PCR) using primers designed for c-MYC exon2aswell CD21+weak,CD22+,CD23-, and HLA-DR+, as determined by as the 4 constant region genes of IgH.9 Fig. 5A shows the two-color flow cytometry (Fig. 2). These cells expressed the results ofLD-PCR with a combination ofthe c- MYC and a µandd heavy chains in association with the l light chain on constant gene primers. The LD-PCR product, which was 5.4 their surfaces (Fig. 2). The DNA index compared with that kb in size, was cloned into the plasmid (pGEM® -T Easy, ofnormal diploid cells was 1.00. A bone marrow core biopsy Promega), and three independent cloned DNAs were se- sample was normocellular with interstitial infiltrates of quenced using an ABI 310 automated sequencer (Applied intermediate-sized lymphoid cells and normal trilineage he- Biosystems) in order to avoid PCR artifacts. The nucleotide matopoiesis. Immunohistochemistry confirmed that the infil- sequences ofthe regions ofinterest were analyzed via the trated cells were positive for CD5 and CD20, and negative for University of California Santa Cruz Genome Bioinformatics CD3 and cyclin D1. In situ hybridization for Epstein-Barr database using BLAT (genome.ucsc.edu/cgi-bin/hgBlat/) in virus-encoded RNA was negative. order to determine the sequence ofthe c- MYC-IgH junction, in which breakage and reunion occurred within the non- coding region ofc- MYC exon 1 and the a switch region of Cytogenetic and molecular analyses IgH (Fig. 5B). G-banding ofmetaphase spreads obtained fromthe bone marrow revealed two types ofaneuploid cell, both ofwhich Treatment course had a 14q + marker chromosome. We next performed fluo- rescence in situ hybridization (FISH) ofthe bone marrow Although the patient’s condition had been good for sev- smear slide using the c-MYC and immunoglobulin heavy- eral months, his hemoglobin level gradually fell to 9.1 g/dL

Fig. 1. Appearance ofleukemia cells in the peripheral blood ( 1A) and bone marrow (1B). These cells were characterized by moderately condensed nuclear chromatin, prominent nucleoli, and a faintly basophilic cytoplasm. Wright staining; original magnification, × 100 objective.

220 t(8;14) B-PLL with AIHA and PRCA

Fig. 2. Flow cytometry ofleukemia cells. Gated cells on the SSC-FSC scattergram were sepa- rated by the expression ofthe antigens indicated. These cells were positive forCD5, CD19, CD20, and HLA-DR, but were negative for CD10 and CD23. Cell surface immunoglobulins were the µ and d heavy and l light chains.

Fig. 3. Fluorescence in situ hybridization (FISH) showing the c-MYC-IgH fusion gene and t(8;14)(q24; q32). (3A) FISH ofthe bone marrow smear slide. Round nuclei corresponding to prolymphocytes contain one green (IgH), one red (c-MYC), and two yellow (c-MYC-IgH fusion) signals, as indicated by the arrows ofeach color. A lobulated nucleus corresponding to myeloid cells carries two green and two red signals (asterisk). The blue signals represent the centromere region ofchromosome 8 (CEP8). ( 2B) Metaphase FISH showing the fusion signals on the der(8) and der(14) chromosomes (yellow arrows). One IgH signal was localized on normal chromosome 14 (green arrow) and one c-MYC signal was on der(3)t(3;8)(p13;q13) (red arrow). The G-banded picture is shown on the right as a reference. The karyotype of this metaphase chromosome was 47,XY,+der(3)t(3;8)(p13;q13),-8,t(8;14)(q24;q32),+18.

221 Iioka F, et al.

Fig. 4. The G-banded karyotype obtained from the bone marrow. The karyotype was 48,XY,+add(3)(p13),t(8;14)(q24;q32),+18. The arrows indicate add(3)(p13), der(8)t(8;14) (q24;q32), and der(14)t(8;14)(q24;q32) chromosomes and +18.

IgH α

Fig. 5. Long-distance polymerase chain reaction (LD-PCR) showing the c-MYC-IgH fusion gene. (5A) Ethidium bromide-stained gel electrophoresis ofLD-PCR products. Lane M, 1-kb ladder as a molecular weight marker; and lane 1, peripheral blood ofthis case. The primers were: MYC/04, 5'-ACAGTCCTGGATGATGATGTTTTTGATGAAGGTCT-3'; and IGHA/01, 5'-TTCGTGTAGTGCTTCACGTGGCATGTCACGGACTT-3'.9 (5B) Nucleotide sequences encompassing the t(8;14)(q24;q32)/c-MYC-IgH junction. A closed arrowhead indicates the breakpoint within c-MYC exon 1, which was located 320 bp upstream ofthe exon 1-intron 1 boundary ofc- MYC. An 8-bp segment ofunknown origin was inserted at the junction.

(Fig. 6). Both direct and indirect Coombs’ tests were positive ng/mL; lactate dehydrogenase, 382 IU/L; and total bilirubin, and his haptoglobin value was 1.3 mg/dL (normal range, 9- 1.4 mg/dL. Marked erythroid hypoplasia was observed in the 40 mg/dL). Nine months after his initial presentation, he was bone marrow and comprised 0.6% ofthe nucleated cells, admitted to our department because ofincreasing dyspnea on while myeloid lineage cells and megakaryocytes were within exertion. On examination, he appeared to be markedly ane- adequate ranges. The extent ofprolymphocyte infiltration mic, but did not exhibit hepatosplenomegaly or lymphadenop- was not significantly different from that in the initial exami- athy. Laboratory tests revealed the following: hemoglobin nation. Serological tests for parvovirus B19 IgM and the level, 5.4 g/dL; white cell count, 16.7 × 103/µL, including DNA ofthis virus were negative. 63.5% prolymphocytes; and platelet count, 202 × 103 /µL. The patient was initially treated with prednisolone, which The proportion ofreticulocytes was 0.1% ofhis red blood led to a burst ofreticulocytes and the deterioration ofperiph- cells (3,660/µL); serum iron level, 251 µg/dL; ferritin, 572 eral prolymphocytosis (Fig. 6). He then received 4 weekly

222 t(8;14) B-PLL with AIHA and PRCA

Fig. 6. Clinical course and response to treatment. The treatment consisted ofpredniso- lone, which was started at a dose of60 mg/day and tapered as illustrated, and weekly doses of375 mg/m 2 rituximab. The counts ofwhite cells (WBC) and prolymphocytes in the peripheral blood, level ofhemoglobin (Hb), and percentage ofreticulocytes are shown. doses of375 mg/m 2 rituximab; we divided the first dose into c-MYC oncoprotein, which is a transcriptional factor that 50 mg/m2 on day 1 and 325 mg/m2 on day 3 to prevent an activates genes involved in cell proliferation and growth, infusion reaction. As shown in Fig. 6, prolymphocytes tumors carrying the translocation generally exhibit aggressive quickly disappeared from the peripheral blood and his hemo- histopathological and clinical behaviors.15 In B-CLL, the globin level steadily increased. Other laboratory data related presence oft(8;14)(q24;q32) indicates cytological and clinical to AIHA and PRCA were within normal ranges. His condi- transformations to an aggressive disease as well as a poor tion is currently good, having achieved complete remission clinical outcome.16 On the other hand, a previous study de- (CR) of the underlying leukemia 40 months after his first scribed 9 cases ofB-PLL that carried the c- MYC translocation referral to our department. determined by FISH; the median time to treatment was as short as 1.5 months, 5 ofthe 9 cases achieved CR or a partial response by the initial treatment, and the median overall sur- DISCUSSION vival from the clinical diagnosis was extended to 110 We herein describe a case ofB-PLL carrying t(8;14)(q24; months.17 In the present case, B-PLL itselfexhibited indolent q32)/c-MYC-IgH, which was determined by both G-banding clinical behavior and responded well to rituximab, which led and interphase/metaphase FISH. LD-PCR and nucleotide se- to CR. Thus, the role oft(8;14)(q24;q32)/c- MYC-IgH in the quencing analyses confirmed the c-MYC-IgH rearrangement, pathogenesis of de novo B-PLL may not be identical to that in in which the positions ofthe breakpoints on both c- MYC and aggressive lymphoid neoplasms. A very speculative mecha- IgH were similar to those ofsporadic-type Burkitt nism for this is that high-level expression of c-MYC targets lymphoma.9 The patient did not exhibit the typical features of genes and collaborating genetic mutations,18,19 both ofwhich B-PLL; namely, splenomegaly was absent and only a modest effectively separate Burkitt lymphoma from diffuse large B- increase was observed in the peripheral white cell count. cell lymphoma and are likely to correlate with the aggressive Nevertheless, the characteristic morphology ofleukemic cells behavior, which may not be the case in B-PLL carrying t(8;14) and strong expression ofsurfaceimmunoglobulins and B-cell (q24;q32)/c-MYC-IgH. antigens favored the diagnosis of B-PLL. The patient developed progressive anemia and finally the Only a few studies have involved cytogenetic analyses of aplastic crisis oferythroid precursors. Laboratory tests and a B-PLL due to the rarity of this disease and difficulties associ- bone marrow examination suggested that the anemia resulted ated with examining metaphase chromosomes.3 from mechanisms mediating both AIHA and PRCA; that is, Nevertheless, cases ofB-PLL that carry t(8;14)(q24;q32) have positive antiglobulin tests and a low haptoglobin level indi- been sporadically reported in the literature.10-14 As t(8;14) cated the former, while reticulocytopenia in the peripheral (q24;q32)/c-MYC-IgH leads to deregulated expression ofthe blood and restricted erythroid hypoplasia in the bone marrow

223 Iioka F, et al. supported the latter mechanism. Hirokawa et al. identified a al.: Acquired pure red cell aplasia associated with malignant lym- total of22 patients with lymphoma-associated PRCA world- phomas: a nationwide cohort study in Japan for the PRCA wide in the literature between 1978 and 2007, 7 ofwhom had Collaborative Study Group. Am J Hematol 84:144-148, 2009 a positive Coombs’ test.7 Choi et al. described an additional 8 ISCN (2009): An International System for Human Cytogenetic case ofangioimmunoblastic T-cell lymphoma associated with Nomenclature (2009). Basel, S. Karger, 2009 PRCA and a positive Coombs’ test, both ofwhich were re- 9 Akasaka T, Muramatsu M, Ohno H, Miura I, Tatsumi E, et al.: solved with CHOP chemotherapy together with the underly- Application oflong-distance polymerase chain reaction to detec- ing lymphoma.20 These findings suggest that autoantibodies tion ofjunctional sequences created by chromosomal translocation targeting the differentiation stages of both mature erythro- in mature B-cell neoplasms. Blood 88:985-994, 1996 cytes and erythroid precursors can be concurrently produced 10 Crisostomo RH, Fernandez JA, Caceres W: Complex karyotype in the setting oflymphoid tumors. In the present case, rituxi- including chromosomal translocation (8;14)(q24;q32) in one case mab appeared to have exerted its effects not only on neoplas- with B-cell prolymphocytic leukemia. Leuk Res 31:699-701, 2007 tic B-PLL cells, which strongly expressed CD20, but also on 11 Kuriakose P, Perveen N, Maeda K, Wiktor A, Van Dyke DL: polyclonal antibody-producing B cells, leading to the resolu- Translocation (8;14)(q24;q32) as the sole cytogenetic abnormality tion ofanemia and long-term CR ofthe underlying B-PLL. in B-cell prolymphocytic leukemia. Cancer Genet Cytogenet 150: 156-158, 2004 12 Lens D, Coignet LJ, Brito-Babapulle V, Lima CS, Matutes E,et CONFLICT OF INTEREST al.: prolymphocytic leukaemia (B-PLL) with complex kar- The authors declare no conflict of interest. yotype and concurrent abnormalities ofthe p53 and c-MYC gene. Leukemia 13:873-876, 1999 13 Merchant S, Schlette E, Sanger W, Lai R, Medeiros LJ: Mature REFERENCES B-cell leukemias with more than 55% prolymphocytes: report of2 1 Campo E, Catovsky D, Montserrat E, Müller-Hermelink HK, cases with Burkitt lymphoma-type chromosomal translocations Harris NL, et al.: B-cell prolymphocytic leukaemia. In: involving c-myc. Arch Pathol Lab Med 127:305-309, 2003 Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, et al. 14 Flatley E, Chen AI, Zhao X, Jaffe ES, Dunlap JB, et al.: (eds): World Health Organization Classification of Tumours, Aberrations of MYC are a common event in B-cell prolymphocytic WHO ClassificationofTumours ofHaematopoietic and leukemia. Am J Clin Pathol 142:347-354, 2014 Lymphoid Tissues. 4th ed, Lyon, International Agency for 15 Klapproth K, Wirth T: Advances in the understanding ofMYC- Research on Cancer (IARC), pp.265-266, 2008 induced lymphomagenesis. Br J Haematol 149:484-497, 2010 2 Dungarwalla M, Matutes E, Dearden CE: Prolymphocytic leukae- 16 Huh YO, Lin KI, Vega F, Schlette E, Yin CC, et al.: MYC mia ofB- and T-cell subtype: a state-of-the-art paper. Eur J translocation in chronic lymphocytic leukaemia is associated with Haematol 80:469-476, 2008 increased prolymphocytes and a poor prognosis. Br J Haematol 3 Dearden C: How I treat prolymphocytic leukemia. Blood 120: 142:36-44, 2008 538-551, 2012 17 Put N, Van Roosbroeck K, Konings P, Meeus P, Brusselmans C, 4 Hauswirth AW, Skrabs C, Schützinger C, Gaiger A, Lechner K, et et al.: Chronic lymphocytic leukemia and prolymphocytic leuke- al.: Autoimmune hemolytic anemias, Evans’ syndromes, and pure mia with MYC translocations: a subgroup with an aggressive red cell aplasia in non-Hodgkin lymphomas. Leuk Lymphoma 48: disease course. Ann Hematol 91:863-873, 2012 1139-1149, 2007 18 Love C, Sun Z, Jima D, Li G, Zhang J, et al.: The genetic land- 5 Zent CS, Kay NE: Autoimmune complications in chronic lym- scape ofmutations in Burkitt lymphoma. Nat Genet 44:1321- phocytic leukaemia (CLL). Best Pract Res Clin Haematol 23:47- 1325, 2012 59, 2010 19 Dave SS, Fu K, Wright GW, Lam LT, Kluin P, et al.: Molecular 6 Vlachaki E, Diamantidis MD, Klonizakis P, Haralambidou- diagnosis ofBurkitt’s lymphoma. N Engl J Med 354:2431-2442, Vranitsa S, Ioannidou-Papagiannaki E, et al.: Pure red cell aplasia 2006 and lymphoproliferative disorders: an infrequent association. 20 Choi JH, Oh YH, Park IK: A case ofpure red cell aplasia associ- ScientificWorldJournal 2012:475313, 2012 ated with angioimmunoblastic T-cell lymphoma. Cancer Res Treat 7 Hirokawa M, Sawada K, Fujishima N, Kawano F, Kimura A, et 42:115-117, 2012

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